Figure 3. ESX-1 activity is required for presentation of EsxA_28-36 and EsxJKPW_24-34 on MHC-I, independently of type I interferon signaling.

(A) Schematic representation of the localization of EsxA and EsxJKPW in macrophages infected with wild-type Mtb H37Rv or the ESX-1-deficient eccCa1:Tn transposon mutant. (B) Schematic showing our workflow for targeted detection and quantification of Mtb-derived MHC-I peptides by SureQuant, using stable isotope labeled peptide-MHC complexes (hipMHCs) as internal standards. SIL: stable isotope labeled. (C) Relative quantification of EsxA_28-36 and EsxJKPW_24-34 by SureQuant in macrophages infected with no Mtb (mock), wild-type Mtb H37Rv, or eccCa1:Tn for n=3 donors (all HLA-A*02:01+, HLA-B*57:01+). As oxidation of methionine is common during sample handling, both the oxidized and non-oxidized form of EsxJKPW_24-34 were quantified. (D) Luminescence as a function of time measured for macrophages infected with luciferase-expressing Mtb, in a wild-type H37Rv or eccCa1:Tn background, with or without the addition of 10 ng/mL IFN-β in the culture media. Addition of 25 µg/mL rifampicin (RIF) to the culture media was used as a control showing reduced luminescence with bacterial death. Data points and error bars represent the mean and standard deviation of n=3 donors, each of which represents the mean of three technical replicates. (* p<0.05, one-way ANOVA with Dunnett’s multiple comparisons test, relative to H37Rv as the reference condition). (E) CXCL10 concentration in the culture media 72 hr post-infection quantified by ELISA. Data points each represent the mean of three technical replicates for a given donor. (* p<0.05, ** p<0.01, one-way ANOVA with Tukey’s multiple comparisons test on log-transformed concentrations.) (f) Relative quantification of EsxA_28-36 and EsxJKPW_24-34 by SureQuant in macrophages infected with no Mtb (mock), wild-type Mtb H37Rv, or eccCa1:Tn for n=3 donors (all HLA-A*02:01+, HLA-B*57:01+). (**** p<0.001, one-way ANOVA with Tukey’s multiple comparisons test.). Error bars indicate standard deviation.

Figure 3—figure supplement 1. Infection of macrophages with wild-type or ESX-1-deficient Mtb strains results in similar rates of infection and does not affect surface MHC-I levels 72 hr post-infection.

Macrophages infected with GFP-expressing wild-type (H37Rv) or ESX-1-deficient (eccCa1:Tn) Mtb were surface stained for MHC-I 72 hr post-infection and analyzed by flow cytometry. (A) Representative contour plots of GFP fluorescence intensity vs. autofluorescence, showing gating for GFP +infected cells. (B) Proportion of GFP + cells for n=3 donors (** p<0.01, *** p<0.001; one-way ANOVA with Tukey’s multiple comparisons test). (C) Representative histograms of surface MHC-I fluorescence intensity. (D) Mean MHC-I fluorescence intensity (MFI) for n=3 donors (p-values determined by one-way ANOVA with Tukey’s multiple comparisons test). Error bars indicate standard deviation.